Implant of a safety switch in iPSCs derived neural stem cells for spinal cord repair Pengzhe (Paul) Lu, Daniel Gibbs, and Mark Tuszynski Summary Neural stem cells (NSCs) derived from induced pluripotent stem cells (iPSCs) have great potential for the treatment of neurological disorders, including spinal cord injury (SCI), since they can be derived from adult somatic cells and can be transplanted autologous to avoid ethical concerns of embryo/fetal tissue usage and immunorejection. However, they can form teratoma or tumor after transplantation into the host central nervous system (CNS) due to the persistence of undifferentiated stem cells or spontaneous reactivation of transgenes or genetic instability of adult cells. In addition, prolonged gliogenesis from human NSCs generates large numbers of new glia that migrate into the host, which may be harmful to the host. Therefore, a new strategy is needed to address these concerns by specifically eliminating those proliferating grafted cells at certain time- points post-transplantation for prevention of graft expansion and tumor formation. The proposed study is to implement a safety switch in human iPSCs by knock-in HSV- thymidine kinase (HSV-TK) suicide gene using a newly developed gene editing technology, the CAS9-CRISPR (clustered regularly interspaced short palindromic repeats) system. We will then differentiate iPSCs into NSCs for transplantation into a rat model of SCI. We will subsequently administer the pro-drug, ganciclovir (GCV), at specific time points to optimize NSC graft survival and fill of lesion sites, while eliminating potentially unsafe dividing cells. We hypothesize that GCV treatment will eliminate all dividing grafted human cells to prevent graft expansion and tumor formation, but un-harm differentiated grafted neural cells in the graft, permitting safe and effective use of promising iPSC technology for SCI. The Specific Aim1 is to use the CRISPR system to knock-in HSV-TK into a human iPSCs line, differentiate iPSC-TK into NSC-TK for test in vitro efficiency. The Specific Aim 2 is to determine the minimal dosage, duration and the optimal start time of GCV treatment post-transplantation in SCI model for elimination of dividing grafted cells for prevention of graft expansion and tumor formation. If the graft expansion and tumor formation can be successfully controlled after transplantation of NSCs into SCI site, our strategy could bring human iPSCs derived NSC therapy for spinal cord injury and the treatment of other neurological disorders and diseases closer to clinical application.